Yersinia pestis, the causative agent of plague, has a multicomponent virulence property called the low-Ca2+ response. This consists of at least 13 virulence genes and at least 8 lcr loci that are thought to constitute a regulatory cascade. The lcr loci cause the virulence genes to be regulated in response to environmental cues of Ca2+, nucleotides, and temperature. Long- term goals are to understand the pathway for regulation of virulence genes in the low-Ca2+ response and to learn the mechanisms of action of the virulence proteins. Aim 1: lcrB, C, and D will be characterized for their DNA sequences and protein products. Their possible regulatory functions will be determined from sequence analysis and from effects that specific mutations have an RNA expression from other low-Ca2+ response genes. Aim 2: Y. pestis mutants will be made, that are deficient in expression of LcrG and V antigen. These proteins are encoded by a low-Ca2+ response operon that has a central role in both regulation and virulence. Testable hypotheses will be developed for the functions of these proteins from each mutant's expression of lcr and virulence genes and from its virulence, growth, and elicitation of pathology in mice. Aim 3: 4 lcr gene products will be tested for their ability to function as transcriptional regulators (DNA-binding proteins or modulators of the specificity of RNA polymerase). Aim 4: Two low-Ca2+ response-regulated outer membrane proteins called Yops will be tested for ability to serve as protective antigens and for their localization in infected macrophages and mouse tissues. YopM will be tested for its role as a virulence factor that affects platelet aggregation, and YopE will be tested for its effect on cellular natural defense mechanisms. These data will provide insight on how Y. pestis regulates expression of virulence factors in response to environmental cues and how the low-Ca2+ response functions in the pathogenesis of plague. This insight will facilitate the studies of virulence gene regulation and virulence mechanisms in other important human pathogens.